Sensitivity of HincII to CpG methylation.

Restriction Fragment Length Polymorphism (RFLP) analysis in human genetics can be confounded by sensitivity of restriction enzymes to the cytosine methylation that occurs at many cytosine-guanine (CpG) dinucleotides. Previous reports provided conflicting information on the sensitivity of Hindi to C methylation (1-3). We identified alleles at a chromosome 4pl6 locus that were inherited in a non-Mendelian manner due to inhibition of Hindi cleavage by CpG methylation. We confirmed that cleavage by Hindi, whose recognition sequence is 5' GTPyPuAC 3', is inhibited by the presence of 5-methylcytosine at the terminal base of its recognition site. To assay for methylation sensitivity of Hindi, we used CpG Methylase, which methylates DNA de novo at CpGs (4). We treated two plasmids of known sequence that contain Hindi sites with 24 units of CpG Methylase//tg DNA in 140 /il overnight at 37°C. Then 0.5 /tg of methylated or unmethylated plasmid DNA was digested overnight with 20 units of Hindi in 35 /tl. This level of overtreatment is many-fold greater than is commonly used for genomic DNA digestion. Plasmid 1 contains 5 HincII sites, none of which contain an internal CpG, but two of which are followed by a G, so that the terminal C of the site is part of a CpG. One Hindi site in Plasmid 1 followed by a G was not cut, while the other site was cut partially. The other three sites, which do not contain or overlap with CpGs, were cut to completion (Figure 1). We also tested plasmid Clone B, which contains a Hindi site of sequence 5' AGTTAACG 3' from the HOX7 gene (5) and a site in the vector that contains an internal CpG, but is not followed by a G, allowing us to test whether Hindi digestion is inhibited by methylation of the two Cs in the internal CpG. Cleavage at only one of the two sites in Clone B was inhibited by methylation (Figure 1). Double digests indicated that methylation inhibited cleavage at the Hindi site that is followed by a G, but not at the site with the internal CpG (data not shown).